The present invention relates to the identification of centromeres that are useful, for example, in constructing artificial chromosomes and cells comprising such artificial chromosomes.
Genetic transformation of biological organisms is essential for genetic studies and for construction of novel strains used in biotechnology. There are two general ways of adding genes into the genome of a biological organism: the introduced gene(s) can be integrated into the organism's chromosome(s) or the introduced gene(s) can reside on a new, artificial chromosome that exists autonomously in the genome, independent of the existing chromosomes. If available, artificial chromosomes are generally the vehicles of choice for transformation of eukaryotic organisms, due to a number of reasons, among them: single copy number, stable and autonomous inheritance, lack of disruption of the existing chromosomes, the ability to transfer many genes on a single construct, and high transformation efficiency. As a result, extensive efforts have been directed into construction and testing of artificial chromosomes for transformation of eukaryotes.
The centromere is an important element in an artificial chromosome, mediating faithful chromosome segregation between the two daughter cells in a cell division. Accordingly, the isolation and identification of functional centromere sequences is an essential part of constructing artificial chromosomes for any specific organism. Eukaryotic centromeres vary greatly in size, ranging from 120-200 bp in budding yeasts to tens of megabases in plants and animals. They are also very diverse in structure and sequence, with centromeres in higher eukaryotes often composed of large tracts of tandem satellite repeats, interspersed with retrotransposons and other sequences, including in some cases functional genes. De novo centromere function (i.e., establishment of centromere function from naked DNA introduced into a cell) often requires the specific centromere sequences present in that organism, as sequences from a related organism may not work efficiently in establishing centromere function. The high amount of species specificity of centromere sequences correlates with the observation that centromere sequences evolve very rapidly and can lose all homology between related species within several million years of evolution (e.g., centromere repeat sequences within the genus Arabidopsis). As a result, it is generally not possible to use homology to centromere sequences from a related organism as a method for isolating centromeres from an organism where the centromere has not previously been characterized.
Identification of centromeres in organisms has been pursued in several organisms by searching for repetitive DNA or methylated DNA followed by labeling studies to determine whether the identified sequences hybridize to the centromere region of chromosomes, and/or functional studies to determine whether the identified sequence(s) function as centromeres (see, for example, U.S. Pat. No. 7,456,013, WO 08/112,972).
However, conserved centromere features other than sequence can be exploited to isolate centromere sequences from novel species. For example, CenH3 (known as CENP-A in humans) is a variant of the nucleosome protein histone H3 that is preferentially associated with centromeric chromatin. This protein differs from histone H3 in having longer and divergent N-terminal sequences. Antibodies raised against the unique N-terminal sequences of CenH3 have been used in some strategies for isolating centromere sequences from some species, for example, using chromatin immunoprecipitation (“ChIP”). Because immunoprecipitation of chromatin typically results in isolation of non-specific sequences as well as the sequence(s) of interest, when used for centromere identification, it has been performed in conjunction with hybridization or sequence comparisons with sequence motifs previously known to be associated or suspected of being associated with centromeres in the organism of interest (see, for example, Nagaki et al. Genetics 163: 1221-1225 (2003); Lee et al. Proceedings Natl. Acad. Sci. USA 102: 11793-11798 (2005)), thus relying on prior knowledge of centromere-associated sequences. Thus, there remains a need in the art for methods of identification of centromere sequences that can quickly process and specifically identify centromere sequences (as distinguished from non-specific sequences) among large pools of nucleic acids molecules, when there are no known centromeres for comparison, for example in several algal species where centromere identification has been particularly difficult.